PROJECT SUMMARY/ABSTRACT Specific enhancers interact with promoters to specify the cellular pattern, timing, and levels of gene expression. Enhancers can reside up to megabases away from their target gene promoters and strongly activate transcription. Aim 1 will characterize active enhancer elements and their relationship to promoter elements in vivo in human K562 (a tier 1 ENCODE cell line) by testing a broad array of Transcription Regulatory Elements (TREs) for their enhancer activity using eSTARR-seq, our modified element-clone- compatible STARR-seq assay. This collection of TREs will be selected based on a variety of criteria established by ENCODE and others. Large numbers of selected TREs can be handled using our new Clone- seq method, and then tested for enhancer activity by eSTARR-seq. For the TREs that have significant enhancer activity, ~10,000 synthetic mutations will be generated that are designed to destroy distinct TF binding motifs found within each enhancer. We will generate mutant clones using our en masse Clone-seq2 method and examine their impact on enhancer activity using eSTARR-seq. These data will be used to understand the underlying molecular architecture and function of enhancers and promoters. Aim 2 generates K562 cell lines using CRISPR/Cas9 that contain critical synthetic enhancer mutations identified in Aim 1. PRO- seq assays can then be used to measure with high sensitivity and resolution the transcription at the variant enhancers as well as all TREs and transcription units genome-wide. This will reveal the role of DNA sequence motifs within native enhancer loci in the regulatory crosstalk with distal gene promoters and enhancers. Circularized Chromosome Conformation Capture (4C) experiments with particular enhancers as the anchor site will provide an unbiased analysis of distal interactions, while targeted ChIP-qPCR experiments will test effects of these mutant enhancers on transcription factor binding and local histone marks at these genomic points of enhancer interaction. Thus, Aim 2 rigorously characterizes mutated enhancers from Aim 1 in their native chromatin environment. Aim 3 characterizes the de novo activation of enhancers, which are known to be triggered by the heat shock activation of HSF1, a master regulator. Because the sequence motif, HSE, to which HSF1 binds is well defined, targeted HSE mutations that cripple the enhancer activity will be made immediately using CRISPR at native loci and the effects on transcription genome-wide can be analyzed directly by PRO-seq. Additional critical motifs in these inducible enhancers will be identified in a less biased way by the more laborious, but high-throughput, eSTARR-seq approach described in Aim 1. Finally, tracking the kinetics with which the structural characteristics of these enhancers form in the minutes following heat shock relative to the induced transcriptional activity as measured by PRO-seq allows assessment of which characteristics (DNase I hypersensitivity, histone modifications, binding of HSF1 and other TFs, and eRNA production) correlate with functional transcription effects on distal promoters and other enhancers. !